DESCRIPTION: The effort of the principal investigator is directed toward an understanding of the mechanism by which Phi 6 generates recombinant RNA genomes. RNA recombination is an important mechanism both for repair of the genome and for genome evolution. The Phi 6 recombination is heterologous or "illegitimate"; intergenomic recombinants are generated involving sequences with little identity in and around the site of recombination. RNA recombination does not result from "cutting and joining" of two parents. Rather, according to the current working model, recombination is mediated by the so-called "copy choice" mechanism. The 3' segment of the nascent RNA chain copied from one template jumps onto or captures a new template upon which it is extended further, hence generating a recombinant RNA strand. A procapsid assembled upon translation of genomic transcripts packages single stranded genomic RNAs and subsequently replicates them. The template switching is thought to occur during the synthesis of the minus strand within the capsid. Obviously, an impediment to replication may trigger this recombination event. Thus, the particular recombination phenomenon is a problem of polymerase structure-function-mechanism, rather than that of a classic recombinase. The applicant laboratory has developed an in vitro system for packaging and replication. In this assay, an empty procapsid is preassembled in vivo with plasmid encoded viral proteins. The purified procapsid can package SS RNAs and replicate them to the ds RNA genome. It is this in vitro system that is modified for the study of recombination through the use of genetically engineered RNA molecules. The recombinants are monitored through plaque formation, RT PCR or both. The proposed experiments will continue to investigate the sequence and structural requirements for recombination, and they will begin to decipher the roles of specific procapsid proteins in the recombination process. The experiments will make use of the in vitro system, specific RNA sequences which will be designed, and also mutant forms of the procapsid proteins which are either at hand or generated during this project period. Reaction conditions influencing recombination ie. polymerase pausing and template switching will be examined, and the effects of the mutant RNAs and proteins on recombination will be investigated in vivo to shed light on the recombination mechanism.